The main protagonist of Friendship Is Magic is Twilight Sparkle, a unicorn pony with a light violet body and an indigo mane and tail with pink and purple streaks, and represents the element of Magic. She is Princess Celestia's dearest pupil and initially considers the pursuit of knowledge to be more important than socialization. In the pilot episode, Princess Celestia orders her to take a break from her studies and visit Ponyville to learn the importance of friendship. This proves to be the key to unlocking the "Elements of Harmony" and defeating Nightmare Moon. She decides to stay in Ponyville and study friendship with her mentor's blessings. She is voiced by Tara Strong, and Rebecca Shoichet provides Twilight's singing voice.

She is accompanied by a baby dragon named Spike, who acts as her research assistant, workhorse, closest friend and messenger. He can send written messages to Princess Celestia by burning them with his magical fire breath, and receive her responses by coughing up the scrolls on which they are written. He is often sarcastic and laughs at others' misfortune, but is very supportive towards Twilight and also helpful toward others. He has a huge crush on Rarity, the beauty of the group, but she never seems to notice his passion for her.